Loss of selectivity and increased permeability of the intestinal tight junction barrier contributes to inflammation and diarrhea. An impediment to interpreting and correcting these pathologic changes is that we lack an understanding of the molecular structure of the barrier and the basis for its size- and charge- selectivity and permeability. Recent data support the existence of two distinct pathways through the junction: one is formed by small size- and charge-selective pores;the second allows bulk size-independent flow of larger solutes and is likely to be an important component of pathologic "leakiness". Our long range goal is to understand how specific protein components of the tight junction define selectivity and permeability of these distinct pathways so that the intestinal barrier can be manipulated for therapeutic purposes, with the central hypothesis that specific transmembrane tight junction and adhesive proteins control different aspects of selectivity and permeability. Strong Preliminary Studies support this hypothesis, which will be tested in three Specific Aims. (1) Do claudins, occludin and tricellulin determine the number and size of the small paracellular pores? Based on our published and Preliminary Studies we hypothesize that claudins create the pores and may confer different pore sizes and numbers;other adhesion proteins like occludin may control porosity. The role of these proteins in regulating permselectivity will be tested in overexpression, mutational and siRNA knockdown studies in cultured Caco-2 and other epithelial monolayers. (2) Is the size-independent pathway influenced by non-claudin adhesion molecules at baseline and during physiologic changes? Our working hypothesis is that effectiveness of the size-independent pathway is controlled by adhesion molecules like occludin, tricellulin, JAM-A and other members of the Ig- super family. The role of these proteins will be tested after knockdown and after permeability enhancement by physiologic stimuli in cultured epithelial models (i.e. activating myosin phosphorylation pathways) and in mouse intestine (in JAM-A null mice). (3) Determine the structural basis for claudin interactions. Our working hypothesis is that claudin monomers interact both within one cell membrane and across the paracellular space to create the pores and the continuous barrier. Experiments will define the amino acid sequence rules for these interactions;and the composition of a 650kDa claudin-containing complex isolated in Blue-Native PAGE. The overall approach is innovative because of a profiling assay which allows quantification of the size and number of pores and level of permeability of the non-restrictive path. The significance of this work is that it will define the contribution of each protein to controlling barrier selectivity and permeability. This information can be used to interpret the role of specific protein changes in disease and to begin to design selective targeted strategies to preserve and restore the intestinal barrier or enhance transmucosal drug delivery. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed at understanding at a molecular level how the intestinal epithelium creates a selective barrier between the bowel contents, which include both nutrients and potentially harmful bacteria and antigens, and the rest of the body. The public health significance of this project is that loss of selectivity and increased permeability contribute to a number of intestinal diseases, including diarrhea and inflammation. The findings will guide strategies to preserve and restore the intestinal barrier or enhance permeability for transmucosal drug delivery.